The synthesis and purification steps were controlled in preparation of thymosin α1 to obtain the product with high-purity.In the solid-phase peptide synthesis (SPPS)process

TBTU/HOBt/DIEA was employed as the coupling reagent.In the sites where α-helix or β-turn structure formed easily

re-coupling reagent of HOBt/DIC was used to ensure a complete reaction.In the separation and purification process

a multi-step method was employed

in which reversed-phase ion-pair liquid chromatography was used as the main means

combining the advantages of reversed-phase chromatography and ion chromatography.The results showed that through the re-coupling the crude peptide had a purity of 67.2% with the use of TBTU/HOBt/DIEA reagent in SPPS.The purity of the refined thymosin α1 was above 99.5% with a total yield of 17.2% with the reversed-phase ion-pair liquid chromatography.This separation process avoids complex chromatography separation process.The method may be an efficient way for preparation of peptides with high-purity.